Transcriptional regulation of target genes

ABSTRACT

The present invention describes a method of identifying inducible genetic regulatory sequences that can control the transcription of specific gene transcripts. Methods of using inducible genetic regulatory sequences are also discussed. In particular, the genetic regulatory sequences of the present invention can modulate the transcription of a nucleic acid transcript in vivo.

RELATED PATENT APPLICATIONS

[0001] This application claims priority under 35 USC § 119(e) to U.S.Ser. No. provisional patent application No. 60/292,604 filed May 22,2001, which application is herein specifically incorporated by referencein its entirety.

FIELD OF THE INVENTION

[0002] The present invention discloses a method of providingtranscriptional control of specific gene transcripts. One particular usefor such transcriptional control is in gene therapy. Specific geneticregulatory sequences, signaling devices, and peptides that emulatetranscription factors, as well as the methods of using the same are alsoprovided.

BACKGROUND OF THE INVENTION

[0003] During the past decade researchers have begun to lay a solidfoundation for performing gene therapy related procedures. For example,long-term expression of heterologous genes in mammals was demonstratedusing viral vectors engineered to contain tissue specific promoters(U.S. Pat. No. 6,040,172, herein specifically incorporated by referencein its entirety). In addition, a modification of behavior wasdemonstrated in an animal model for human Parkinson's Disease (U.S. Pat.No. 6,180,613, herein specifically incorporated by reference in itsentirety). However, before gene therapy becomes a general medicalpractice, other factors must be addressed. One such factor concerns theregulation of the expression of the genetic transcript and/or theresulting gene product.

[0004] For example, many gene therapy strategies involve the expressionof genes which are likely to cause adverse effects if they are expressedcontinuously. Thus, administering a gene transcript that is incapable ofbeing regulated will oftentimes be unacceptable. In such cases,performing gene therapy with a regulatable gene expression system isnecessary to control the expression of the otherwise therapeutic geneand/or gene product. Presently, regulatable promoters such as themetallothionein promoter, the tetracycline-on, and the tetracycline-offpromoters are available. Unfortunately, such promoters are notwell-suited for gene therapy because they are controlled by inducercompounds that are either toxic or can become toxic with long-term use.Still others are not practical for specific in vivo applications, e.g.,they cannot cross the blood-brain barrier. Generally, the regulatablepromoters that are currently employed in gene therapy strategies havebeen identified/selected for very different purposes and have thereforenot been optimized for the particular role they must play in genetherapy.

[0005] Therefore, there is a need for developing methods for identifyinggenetic regulatory sequences (e.g., a promoter) that will be responsiveto a stimulus in a particular tissue. In addition, there is a need foridentifying stimulants of such genetic regulatory sequences that areboth non-toxic and can readily gain access to the target tissue. Thereis a need for developing vectors that employ such genetic regulatorysequences for use in gene therapy. Further, there is a need to providemethods of performing gene therapy which employ the novel stimulants andgenetic regulatory sequences of the present invention.

SUMMARY OF THE INVENTION

[0006] The present invention provides specific genetic regulatorysequences that can be controlled by specific stimulants in order toregulate the expression of exogenous genes in vivo. The presentinvention further provides methods for selecting such genetic regulatorysequences. In one embodiment the genetic regulatory sequence responds toa stimulus in a particular place and/or at a particular time. Thus, sucha genetic regulatory sequence may be induced in a particular tissueand/or by the administration of a particular stimulus. For any givengene therapy strategy the type of tissue and stimulant is preferablypre-determined prior to its application.

[0007] In one embodiment, the genetic regulatory sequence responds to apulsatile electromagnetic signal (e.g., stimulation). In a particularembodiment, the pulsatile signal can be turned on and off for at least 1minute at a pulse frequency of 1 to 1000 hertz. In a specificembodiment, the pulse frequency is 10 to 400 hertz. In a more specificembodiment, the pulse frequency is 20 to 200 hertz. In an even morespecific embodiment, the pulse frequency is 30 to 150 hertz. In oneembodiment, the current is between 1 microamp and 50 milliamps. In amore specific embodiment, the current is between 10 microamps and 5milliamps. In one embodiment, the voltage intensity is between 1millivolt and 30 volts. In a more specific embodiment, the voltageintensity is between 100 millivolts and 10 volts.

[0008] In one embodiment, the individual pulse is 1 microsecond to 10seconds. In a specific embodiment, the pulse is 20 microseconds to 1second. In a more specific embodiment, the pulse width is 50microseconds to 0.5 second. In an even more specific embodiment, thepulse width is 90 to 180 microseconds.

[0009] Thus, in one aspect of the present invention methods are providedfor identifying a genetic regulatory sequence that is responsive to apulsatile electromagnetic stimulus. One such method comprises insertinga stimulator into a tissue of an animal subject and applying a pulsatilesignal with the stimulator in which at least a part of the tissue isstimulated. A gene is then identified that has either enhanced ordiminished transcription in the part of the tissue stimulated, and agenetic regulatory sequence involved in the enhanced or diminishedtranscription of the gene is selected. This genetic regulatory sequenceis thus identified as being responsive to the pulsatile electromagneticstimulus. In a particular embodiment, the pulsatile electromagneticsignal is provided by a bipolar stimulator. In a related embodiment thepulsatile electromagnetic signal is provided by a monopolar stimulator.

[0010] In a particular embodiment, at least a part of the tissue is notstimulated by the signal of the stimulator and identifying a gene thathas either enhanced or diminished transcription in the part of thetissue stimulated is performed by comparing the transcription of thegenes in the part of the tissue stimulated with that of a part of thetissue that is not stimulated. In another embodiment, a secondstimulator is placed into the part of the tissue that is not stimulated.In a specific embodiment of this type, no pulsatile signal is applied bythe second stimulator. In an alternative embodiment, the entire tissueis stimulated.

[0011] In one embodiment of the invention, the tissue is neural tissue.In a specific embodiment, the neural tissue is brain tissue. In otherembodiments, the tissue is heart tissue, liver tissue, or pancreatictissue. In a particular embodiment of this embodiment, the pancreatictissue contains the insulin producing beta cells from the islets ofLangerhans.

[0012] In one embodiment, the pulsatile electromagnetic signal ismagnetic. In a particular embodiment of this type the pulsatileelectromagnetic signal is a transcranial magnetic stimulation. Inanother embodiment, the pulsatile electromagnetic signal is electrical.

[0013] In a second aspect, the invention features an isolated geneticregulatory sequence that has been identified by performing a method ofthe present invention. In one embodiment, the genetic regulatorysequence is responsive to a pulsatile electromagnetic signal. In aparticular embodiment the pulsatile electromagnetic signal that thegenetic regulatory sequence is responsive to is magnetic. In analternative embodiment, the pulsatile electromagnetic signal that thegenetic regulatory sequence is responsive to is electrical. In a relatedembodiment, the genetic regulatory sequence is responsive to a specificpeptide identified by a method of the present invention.

[0014] The present invention further provides a vector for the in vivoexpression of a gene of interest in a mammalian host cell which containsa gene of interest operatively under control of a genetic regulatorysequence identified by a method of the present invention. In aparticular embodiment, the vector comprises a gene of interestoperatively linked to a transcriptional control region comprising agenetic regulatory sequence that is responsive to a pulsatileelectromagnetic stimulus. In one embodiment, the pulsatileelectromagnetic signal is magnetic. In another embodiment, the pulsatileelectromagnetic signal is electrical. In a particular embodiment, thegenetic regulatory sequence of the transcriptional control region of thevector exhibits tissue specificity. In a specific embodiment, thegenetic regulatory sequence of the transcriptional control region of thevector is obtained from a gene encoding a protein expressed by the cellwhich is selected to be the target cell for the vector.

[0015] In one embodiment, the vector is a replication defective viralvector. In a particular embodiment of this type, the vector is areplication defective herpes simplex virus (HSV). In another embodiment,the vector is a replication defective papillomavirus. In yet anotherembodiment, the vector is a replication defective Epstein Barr virus(EBV). In still another embodiment, the vector is a replicationdefective adenovirus and/or gutless adenovirus. In yet anotherembodiment, the vector is a replication defective adeno-associated virus(AAV). In still another embodiment, the vector is a replicationdefective lentivirus. In yet another embodiment, the vector is areplication defective retrovirus. In a particular embodiment of thistype, the replication defective retrovirus vector is prepared for use inan ex vivo gene therapy protocol.

[0016] In one embodiment, the genetic regulatory sequence of thetranscriptional control region of the vector is obtained from neuraltissue. In a more specific embodiment of this type, the neural tissue isbrain tissue. In other specific embodiment, the genetic regulatorysequence is obtained from heart tissue, liver tissue, or pancreatictissue. In a particular embodiment of this type, the pancreatic tissuecontains the insulin producing beta cells from the islets of Langerhans.

[0017] In a second aspect, the invention provides a non-human mammalianhost transformed with a vector of the present invention. Preferably theexpression of the gene of interest encoded by the vector can bemodulated by applying a pulsatile electromagnetic signal with astimulator. In a particular embodiment the pulsatile electromagneticsignal is magnetic. In an alternative embodiment the pulsatileelectromagnetic signal is electrical. In a preferred embodiment, thestimulator is also present in the non-human mammalian host.

[0018] In a third aspect, the invention features methods of deliveringthe vectors of the present invention to a target tissue of an animalsubject by administering the vector to the target tissue of the animalsubject. Preferably the expression of the gene of interest encoded bythe vector can be modulated by applying a pulsatile electromagneticsignal with a stimulator. In one embodiment, the pulsatileelectromagnetic signal is magnetic. In an alternative embodiment, thepulsatile electromagnetic signal is electrical. In a particularembodiment, the transcription of the gene of interest in the targettissue is stimulated by the electromagnetic signal. In an alternativeembodiment, the transcription of the gene of interest in the targettissue is hindered and/or inhibited by the electromagnetic signal. In aparticular embodiment, the pulsatile electromagnetic signal is providedby a bipolar stimulator. In a related embodiment, the pulsatileelectromagnetic signal is provided by a monopolar stimulator.

[0019] In a fourth aspect, the invention features methods of identifyinga genetic regulatory sequence that is responsive to a peptide. One suchmethod comprises contacting a peptide with a cell and identifying a genethat has either enhanced or diminished transcription. A geneticregulatory sequence involved in the enhanced or diminished transcriptionof the gene is selected. This genetic regulatory sequence is identifiedas being responsive to the peptide. In a preferred embodiment thispeptide is a random generated peptide. In a particular embodiment, thepeptide has the amino acid sequence of SEQ ID NO:1. In a more specificembodiment, the peptide is between 3 and 40 amino acids long. Morespecifically, the peptide is between 5 and 15 amino acids long. Randomlygenerated peptides that can either enhance or diminish transcription ofthe gene through binding to a genetic regulatory sequence are alsoincluded in the invention. Furthermore, genetic regulatory sequencesthat respond to these peptides are also part of the present invention,as are the vectors that comprise such genetic regulatory sequences.

[0020] In a fifth aspect, the invention features methods of regulatingthe expression of a gene of interest in a target tissue of an animalsubject in which a vector of the present invention has beenadministered. One such method comprises applying a pulsatile signal witha stimulator to modulate the transcription of a gene of interest in thetarget tissue. In a particular embodiment of this aspect, the responseto the pulsatile signal by the genetic regulatory sequence of the vectorstimulates the transcription of the gene of interest in the targettissue. In an alternative embodiment, the response to the pulsatilesignal by the genetic regulatory sequence reduces the transcription ofthe gene of interest in the target tissue. In a preferred embodiment ofthis type, the response to the pulsatile signal by the geneticregulatory sequence stops and/or prevents the transcription of the geneof interest in the target tissue.

[0021] In a sixth aspect, the invention features methods of amelioratingsymptoms due to Parkinson's disease. One such method comprisesadministering glutamic acid decarboxylase to the subthalamic nucleus ofa patient having a symptom of Parkinson's disease. In one suchembodiment, the glutamic acid decarboxylase is administered to thesubthalamic nucleus of the patient via a vector. In one particularembodiment, the vector is constructed to comprise a nucleic acidencoding glutamic acid decarboxylase operatively under the control of agenetic regulatory sequence that is stimulated by a pulsatileelectromagnetic signal. A pulsatile electromagnetic signal is appliedwith a stimulator that had been placed into the subthalamic nucleus ofthe patient. The pulsatile electromagnetic signal stimulates thetranscription of the glutamic acid decarboxylase in the subthalamicnucleus which leads to the amelioration of the symptom due toParkinson's disease. In a preferred embodiment the vector is areplication defective viral vector. In a particular embodiment thevector comprises a chicken beta-actin promoter that is operativelylinked to a nucleic acid encoding human glutamic acid decarboxylase (theCBA-GAD65 viral vector).

[0022] In a seventh aspect, the invention features a method ofmodulating the release of a stored compound by a cell. In a preferredembodiment the compound is a small organic molecule. In one suchembodiment, the compound is a hormone and/or a neurotransmitter such asepinephrine, norepinephrine, dopamine, dopa, serotonin and GABA.

[0023] One embodiment of the method comprises administering to an animalsubject (preferably a human) a vector comprising a nucleic acid encodinga protein (preferably an enzyme) operatively under the control of agenetic regulatory sequence. The genetic regulatory sequence isspecifically chosen for its ability to be stimulated by a pulsatileelectromagnetic signal, whereas the protein is specifically chosen forits ability, when expressed, to stimulate the production of a compoundthat is subsequently stored by a cell of the animal subject. Astimulator is then inserted into a tissue of the animal subject suchthat when it is used to apply a specific signal, the genetic regulatorysequence responds. In a particular embodiment exemplified below, thevector and the stimulator are administered to the subthalamic nucleus. Apulsatile electromagnetic signal is then applied with the stimulator,causing the protein to be expressed which thereby stimulates theproduction of the compound, which in turn is stored by a cell. At asubsequent time, a second pulsatile electromagnetic signal is appliedwith the stimulator which then modulates the release of the storedcompound from the cell. In the Example 4 below, the compound is GABA.When the second pulsatile electromagnetic signal is performed at a lowfrequency, the cell is stimulated to increase the release of GABA,whereas when the second pulsatile electromagnetic signal is performed ata high frequency, the cell is stimulated to decrease (and/or block) therelease of GABA.

[0024] Accordingly, the present invention provides genetic regulatorysequences that respond to pulsatile electromagnetic stimulation and arestimulated by pulsatile electromagnetic signals to facilitate thetranscription of a nucleic acid operatively under their control. Furtherprovided is a genetic regulatory sequence that responds to a pulsatileelectromagnetic signal by hindering the transcription of a nucleic acidoperatively under its control or by preventing the transcription of anucleic acid operatively under its control.

[0025] The present invention also provides vectors and replicationdefective vectors that contain nucleic acids that are operatively underthe control of a genetic regulatory sequence identified by a method ofthe present invention.

[0026] Further, the invention provides a method of modulating thetranscription of a selected nuclei acid in vivo, including turning offtranscription, by placing it operatively under the control of a geneticregulatory sequence identified by a method of the present invention.

[0027] Further, the present invention provides a method of performinggene therapy with a defective viral vector comprising a therapeuticnucleic acid that is operatively under the control of a geneticregulatory sequence of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGS. 1-2 show the response of a neuron in the substantia nigrato electrical stimulation of the subthalamic nucleus. A histogram (20 msbins) of spike counts after an electrical stimulation at t=0 is shown.Each trial of the stimulation is used to create the histogram and as alabeled sweep of the graph. FIG. 1 shows that in normal rats there is alarge increase in impulse activity due to subthalamic nucleusstimulation. FIG. 2 shows an inhibition of spontaneous firing of theneuron in the substantia nigra due to subthalamic nucleus stimulation.

[0029] FIGS. 3-4 show the change of extracellular GABA concentration(FIG. 3) and glutamate concentration (FIG. 4) of the substantia nigraand the subthalamic nucleus during subthalamic nucleus stimulation innaive rats and rats transduced with a CBA-GAD65 viral vector. Thestimulation was applied for two different time periods, two minutes andfive minutes for two different groups of rats as shown:

[0030] (i) 10 Hz, at 500 μA for 2 minutes, (labeled ST1); and

[0031] (ii) 10 Hz, at 500 μA for 5 minutes (labeled ST2).

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides methods for controlling thetranscription of heterologous genes in vivo by employing specificgenetic regulatory sequences that can be up and/or down regulated, andpreferably turned off and/or on by specific stimulators. Furtherprovided are specific expression cassettes that include genes ofinterest operably under the control these genetic regulatory sequences,and methods of delivering these expression cassettes to the targetcells. The methods of delivery include using standard transgenictechniques such as transfection and transduction. The expressioncassettes can be included in both viral and non-viral vectors. Thegenetic regulatory sequences can be characterized based on screeninggene expression patterns in a particular tissue following a definedstimulus. In a particular embodiment, the delivery of the expressioncassette is to the nervous system and the genetic regulatory sequencesare selected for their ability to be regulated in the nervous system.

[0033] The invention features a method for identifying geneticregulatory sequences for controlling expression of genes for genetherapy which respond to a physiological stimulus. In one suchembodiment, a physiological stimulus is administered, and messenger RNAis extracted from the cells or tissue. Microarray analysis (see e.g.,U.S. Pat. No. 6,215,894 and U.S. Pat. No. 6,004,755 herein specificallyincorporated by reference in their entirety) can then be performed toidentify genes having increased expression in response to thephysiological stimulus. The genetic regulatory sequences from thesegenes can then be identified and/or isolated (e.g., by PCR or genomiclibrary screening). The genetic regulatory sequences can then beinserted into expression cassettes to control the expression of a geneof interest. The physiological stimulus can then be used to control theexpression of the gene of interest.

[0034] Methods other than microarray analysis may include, but are notlimited to, differential display (see e.g., U.S. Pat. No. 6,045,998 andU.S. Pat. No. 5,599,672, herein specifically incorporated by referencein their entiretiy), subtractive mRNA hybridization (see e.g., U.S. Pat.No. 5,958,738 and U.S. Pat. No. 5,525,471, herein specificallyincorporated by reference in their entirety), peptide arrays,two-dimensional protein gel electrophoresis and microsequencing ofrelevant peptides.

[0035] Stimuli may include, but are not limited to, electricalstimulation, magnetic fields, heat or cold stimuli, peptide or proteininfusion, chemical or drug infusion, ionizing radiation, microwave orultrasound.

[0036] A genetic regulatory sequence (e.g., a promoter or geneticresponse sequence/element) can be selected based upon the response of anendogenous cellular gene in a target tissue to a particular stimulus.Once identified, the genetic regulatory sequence can then be insertedinto a gene therapy vector to control expression of any gene of interestin that target tissue.

[0037] The mammalian nervous system responds to environmental conditionsincluding housing and enrichment paradigms (Young et al. (1999) Nat.Med. 5:448-53 and Rampon et al. (2000) Proc. Natl. Acad. Sci. U.S.A.97:12880-12884), transcranial magnetic stimulation (Fujiki et al. (1997)Brain Res. Mol. Brain Res. 44:301-308; Muller et al. (2000)Neuropsychopharm. 2:205-15; and Hausmann et al. (2000) Brain Res. Mol.Brain Res. 76:355-62) and peripheral administration of exogenouscompounds and peptides by inducing transcription of specific genes.

[0038] The expression of a number of proteins have been found to beresponsive to high frequency TMS in rats, including glial acidicfibrillary protein (GFAP) expression (Fujiki et al. (1997) supra),c-fos, brain-derived neurotropic factor (BDNF) and cholecystokinin (CCK)(Muller et al. (2000) supra, and Hausmann et al. (2000) supra). Thegenetic regulatory equences of these genes may be employed in expressioncassettes by the method of the present invention.

[0039] One particular aspect of the present invention is the use ofessentially any peptide e.g., preferably 3 to 40 amino acid residues,and more preferably 5 to 15 amino acid residues, to selectively upand/or down regulate, and preferably turn on and/or turn off specificgenes. Therefore, the present invention provides methods ofidentifying/mapping genes that are up and/or down regulated, andpreferably turned on and/or off by any given peptide.

[0040] In particular, in Example 3 below, genes encoding the NMDAreceptor, F3 contactin, and MAP-2 are shown to be upregulated inresponse to a novel peptide. Therefore,

[0041] genetic regulatory sequences from the 5′ promoter region of thesegenes are provided as a component of the gene switch. Initially, theisolated promoters can be cloned upstream of a marker gene such as greenfluorescent protein (see e.g., U.S. Pat. No. 5,625,048, WO 97/26333, andWO 99/64592, herein specifically incorporated by reference in theirentirety) or luciferase, which can then be used to further characterizethe responsiveness to the peripheral administration of the peptide.Subsequently, important neural proteins (e.g., tyrosine hydroxylase,DARP-32 etc.) can be inserted in place of the marker proteins for genetherapy, for example.

[0042] Similarly, synthetic promoter constructs including multiplegenetic regulatory sequences from the genes encoding CRE, p53, AP-1,SRE, NF-kappaB, SRF, Spl (using e.g., 3 to 10 copies of the responseelement) can be initially placed upstream of a luciferase and GFP cDNA.These expression cassettes can be cloned into an AAV cis plasmid andused to generate rAAV-RE (generic response element)-luc/GFP, forexample. One such expression vector, the rAAV-Cre-luc has already beenpackaged and successfully injected into a group of rats. The presentinvention further provides “cocktails” of response elements which can beused to generate synthetic response elements that can either upregulatea given transgene or downregulate the transgene, depending on thestimulus applied.

[0043] Vectors may include, but are not limited to, adenovirus(recombinant and “gutless” vectors), herpes simplex virus (recombinantvectors and amplicons), adeno-associated virus, lentivirus, retrovirus,synthetic or non-viral vectors (including liposome and plasmid-basedsystems). Preferably the viral vectors are reproduction defective viralvectors.

[0044] It should also be noted that chimeric promoters or geneticresponse elements which contain some part of an endogenous promoteridentified to be responsive to an external stimulus are also part of thepresent invention.

[0045] Definitions

[0046] As used herein, the term “gene” refers to an assembly ofnucleotides that encodes a polypeptide and includes cDNA and genomic DNAnucleic acids. A gene is a nucleic acid that does not necessarilycorrespond to the naturally occurring gene which contains all of theintrons and regulatory sequences, e.g., promoters, present in thenatural genomic DNA. Rather, a gene encoding a particular protein canminimally contain just the corresponding coding sequence for theprotein.

[0047] As used herein a “promoter sequence” is a DNA regulatory regioncapable of binding RNA polymerase in a cell and initiating transcriptionof a downstream (3′ direction) coding sequence. For purposes of definingthe present invention, the promoter sequence is bounded at its 3′terminus by the transcription initiation site and extends upstream (5′direction) to include the minimum number of bases or elements necessaryto initiate transcription at levels detectable above background. Withinthe promoter sequence will be found a transcription initiation site(conveniently defined for example, by mapping with nuclease S1), as wellas protein binding domains (consensus sequences) responsible for thebinding of RNA polymerase.

[0048] As used herein transcriptional and translational controlsequences are DNA regulatory sequences, such as promoters, enhancers,terminators, and the like, that provide for the expression of a codingsequence in a host cell. In eukaryotic cells, polyadenylation signalsare control sequences.

[0049] An “expression control sequence” is a DNA sequence that controlsand regulates the transcription and translation of another DNA sequence.A coding sequence is “operatively under the control” of transcriptionaland translational control sequences in a cell when RNA polymerasetranscribes the coding sequence into a precursor RNA, which is thentrans-RNA spliced to yield mRNA and translated into the protein encodedby the coding sequence.

[0050] A nucleotide sequence is “operatively under the control” of agenetic regulatory sequence when the genetic regulatory sequencecontrols and/or regulates the transcription of that nucleotide sequence.That genetic regulatory sequence can also be referred to as being“operatively linked” to that nucleotide sequence.

[0051] As used herein, a “genetic regulatory sequence” is a nucleic acidthat: (a) acts in cis to control and/or regulate the transcription of anucleotide sequence, and (b) can be acted upon in trans by a regulatorystimulus to promote and/or inhibit the transcription of the nucleotidesequence. Therefore, an inducible promoter is a genetic regulatorysequence. In addition, a portion of a promoter (e.g., fragment/element)that retains and/or possesses the ability to control and/or regulate thetranscription of a nucleotide sequence either alone or in conjunctionwith an alternative promoter or fragment thereof (e.g., a chimericpromoter) is also a genetic regulatory sequence. Such fragments includeresponse elements (genetic response elements) and promoter elements.

[0052] As used herein, an “expression cassette” is a nucleic acid thatminimally comprises a nucleotide sequence to be transcribed (e.g., acoding sequence) that is operatively under the control of a geneticregulatory sequence.

[0053] A “signal sequence” can be included before the coding sequence.This sequence encodes a signal peptide, N-terminal to the polypeptide,that communicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

[0054] As used herein, a “defective viral vector”, abbreviated “dvv” isa viral vector that requires the expression and/or transcription of atleast one nucleic acid that it lacks in order to be replicated and/orpackaged. Preferably the dvv is a replication defective viral vector.

[0055] As used herein, a “heterologous gene” is a gene that has beenplaced into a vector or cell that does not naturally occur in thatvector or cell.

[0056] As used herein, a gene is an “exogenous gene” when the gene isnot encoded by the particular vector or cell.

[0057] A “vector” as used herein is a genetic construct that facilitatesthe efficient transfer of a nucleic acid (e.g., a gene) to a cell. Theuse of a vector can also facilitate the transcription and/or expressionof that nucleic acid in that cell. Examples of vectors include plasmids,phages, amplicons, viruses and cosmids, to which another DNA segment maybe attached so as to bring about the replication of the attachedsegment.

[0058] As used herein, “pulsatile” stimulation is a stimulation in whichmore than one pulse per unit time (and preferably a series of pulses) isgenerated at a defined pulse width. The number of pulses per unit timeis termed its frequency which can be denoted in Hertz.

[0059] As used herein, a “pulse width” is the length of time a singlepulse lasts.

[0060] As used herein, a “small organic molecule” is an organiccompound, or organic compound complexed with an inorganic compound(e.g., metal) that has a molecular weight of less than 3 kilodaltons,and preferably less than 1.5 kilodaltons.

[0061] Gene Therapy

[0062] The genetic regulatory sequences of the present invention can beused to modulate gene transcription in any cell, including human cells.However, the genetic regulatory sequences of the present invention canbe used to modulate gene transcription in cells of other mammals, suchas rodents, e.g., mice, rats, rabbits, hamsters and guinea pigs; farmanimals e.g., sheep, goats, pigs, horses and cows; domestic pets such ascats and dogs, higher primates such as monkeys, and the great apes suchbaboons, chimpanzees and gorillas.

[0063] The genetic regulatory sequences of the present invention can beoperatively linked to any heterologous nucleic acid of interest,preferably those encoding proteins. Indeed, any protein can be encodedby the heterologous nucleic acid operatively under the control of agenetic regulatory sequence of the present invention. A short list of afew of these proteins and their roles in particular conditionsand/diseases is included in Table 3 below. However, this listing shouldin no way limit the general methodology of the present invention whichprovides the ability to modulate the expression of any nucleic acid ofchoice. In addition, the expression cassettes of the present inventioncan be constructed to comprise multiple nucleic acids each encoding adifferent protein and all under the control of the same geneticregulatory sequence. Alternatively, different nucleic acids can beplaced under the control of different genetic regulatory sequences. Forexample, the use of two genetic regulatory sequences, one of whichstimulates transcription when treated with a pulsatile electromagneticsignal and the other which hinders transcription under the sameconditions can be used to control the expression of two different genesat the same time by operatively linking one coding sequence to onegenetic regulatory sequence and the other coding sequence to the othergenetic regulatory sequence. For example, insulin and glucagonexpression could be controlled in this manner. Alternatively, multipleexpression cassettes can be employed encoding multiple differentproteins. TABLE 3 Genetic Defects Disease/Symptom adenosine deaminasesevere combined immunodeficiency disease alpha, - antitrypsin pulmonaryemphysema  5-alpha reductase male pseudohemaphroditism 17-alphareductase male pseudohemaphroditism p53 or ARF-P19 proteins linked tocancer insulin insulin-dependent diabetes sickle cell anemia B-globinhypoxanthine guanine Lesh-Nyhan Syndrone phosphoribosyl-transferaseornithine transcarbamolase Fatal to newborn males phenylalaninehydroxylase Phenylketonuria Dralassemia x- or B-globin AT Page 7AMenkes' syndrome AT Page 7B Wilson Disease hexosamindase A Tay-SachsDisease acid cholesterylester hydrolase Wolmon Disease

[0064] In one particular example a defective viral vector comprising anucleic acid encoding insulin operatively under the control of a geneticregulatory sequence of the present invention can be employed totransduce the pancreas in vivo to treat insulin-dependent diabetes. Forexample, if the genetic regulatory sequence is induced to stimulatetranscription when a pulsatile electromagnetic signal is provided by astimulator, expression of insulin could be controlled and theinsulin-dependent diabetes treated. The vectors of the present inventioncan be delivered in vitro, ex vivo and in vivo.

[0065] When the genetic regulatory sequence is contained in a viralvector, the delivery can be performed by stereotaxic injection into thebrain for example, as previously exemplified (U.S. Pat. No. 6,180,613,herein specifically incorporated by reference in its entirety); or via aguide catheter (U.S. Pat. No. 6,162,796, herein specificallyincorporated by reference in its entirety) to an artery to treat theheart. In addition, the vectors of the present invention may also bedelivered intravenously, intracerebroventricularly and/or intrathecally,for specific applications. Additional routes of administration can belocal application of the vector under direct visualization, e.g.superficial cortical application, or other non-stereotacticapplications.

[0066] For targeting a vector to a particular type of cell, it may benecessary to associate the vector with a homing agent that bindsspecifically to a surface receptor of the cell. Thus, the vector may beconjugated to a ligand (e.g., enkephalin) for which certain nervoussystem cells have receptors, or a surface specific antibody. Theconjugation may be covalent, e.g., a crosslinking agent such asglutaraldehyde, or noncovalent, e.g., the binding of an avidinatedligand to a biotinylated vector.

[0067] In addition, the helper-free defective viral vectors of thepresent invention can be delivered ex vivo, as exemplified by Andersonet al. (U.S. Pat. No. 5,399,346, herein specifically incorporated byreference in its entirety).

[0068] Alternatively, a vector can be introduced by lipofection.Liposomes can be used for encapsulation and transfection of nucleicacids. Synthetic cationic lipids designed to limit the difficulties anddangers encountered with liposome mediated transfection can be used toprepare liposomes for in vivo transfection of a gene encoding a marker(Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417; see alsoMackey et al. (1988), Proc. Natl. Acad. Sci. U.S.A 85:8027-8031). Theuse of cationic lipids may promote encapsulation of negatively chargednucleic acids, and also promote fusion with negatively charged cellmembranes (Felgner et al. (1989) Science 337:387-388). The use oflipofection to introduce exogenous genes into the specific organs invivo has certain practical advantages. Molecular targeting of liposomesto specific cells represents one area of benefit. It is clear thatdirecting transfection to particular cell types would be particularlyadvantageous in a tissue with cellular heterogeneity, such as pancreas,liver, kidney, and the brain. Lipids may be chemically coupled to othermolecules for the purpose of targeting (Mackey et. al. (1988) supra).

[0069] It is also possible to introduce the vector as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (see, e.g., Wu et al. (1992) J. Biol. Chem.267:963-967; Wu et al. (1988) J. Biol. Chem. 263:14621-14624; Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

[0070] In an ex vivo method of the invention, the genetic regulatorysequences of the invention are delivered to a host cell to betransplanted into a mammalian recipient. The host cells may beendogenous or exogenous to the mammalian recipient. The term “transplantcell” refers broadly to the component, e.g., tissue or cells, beinggrafted, implanted, or transplanted into a recipient subject. As usedherein, the term “transplantation” refers to the transfer or grafting oftissues or cells from one part of a subject to another part of the samesubject or to another subject. Transplanted tissue may comprise acollection of cells of identical composition, or derived from a donororganism, or from an in vitro culture. Delivery of the geneticregulatory sequences of the invention to a transplant cell may beaccomplished by any of the methods known to the art and described above,e.g., as a plasmid, as part of a vector; by injection, lipofection, etc.

[0071] A variety of dissociated cells can be implanted, using standardtechniques for isolation and transplantation of tissue or organs, suchas livers. See, for example, U.S. Pat. No. 6,281,015, hereinspecifically incorporated by reference.

[0072] Transgenic Animals

[0073] A transgenic animal model can be prepared so as to contain anucleic acid operatively under the control of a genetic regulatorysequence of the present invention. For example transgenic vectors,including viral vectors, or cosmid clones (or phage clones) can beconstructed. Cosmids may be introduced into transgenic mice usingpublished procedures (Jaenisch (1988) Science 240:1468-1474).

[0074] Thus the present invention further provides transgenic, knock-in,and knockout animals that contain one or more heterologous genesoperatively under the control of a genetic regulatory sequence of thepresent invention. These animals can be used as animal models in drugscreening assays. In one such example, a drug can be added under various“controlled” expression levels of a particular gene, or at various timepoints before and/or after induced expression of the particular gene,allowing a much more detailed investigation of the effects of that drugon a particular condition. In a specific embodiment, the transgenic,knock-in, or knockout animal is a mouse. Cells from the inducibleknockout, knock-in and/or transgenic animals of the present inventionare also part of the present invention. These cells can also be used inthe drug assays, for example.

[0075] Transgenic animals can be obtained through gene therapytechniques described above or by microinjection of a nucleic acid forexample, into an embryonic stem cell or an animal zygote (such as abacterial artificial chromosome (BAC) comprising a nucleic acidoperatively under the control of a genetic regulatory sequence of thepresent invention). Microinjection of BACs has been shown to besuccessful in a number of animals including rats, rabbits, pigs, goats,sheep, and cows (in Transgenic Animals Generation and Use (1997) ed., L.M. Houdebine, Harwood Academic Publishers, The Netherlands). Methods ofconstructing BACs or other DNAs such as bacteriophage P1 derivedartificial chromosomes (PACs) that encode specific nucleic acids throughhomologous recombination have recently been described in great detail(Heintz et al. (1998) PCT/US98/12966, herein specifically incorporatedby reference in its entirety). Alternatively, a yeast artificialchromosome (YAC) can be used.

[0076] Ribozymes and Antisense

[0077] Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule (seeWeintraub (1990) Sci. Amer. 262:40-46; Marcus-Sekura (1987) Nucl. AcidRes, 15:5749-5763; Marcus-Sekura (1988) Anal. Biochem. 172:289-295);Brysch et al. (1994) Cell Mol. Neurobiol. 14:557-568). Preferably, theantisense molecule employed is complementary to a substantial portion ofthe mRNA. In the cell, the antisense molecule hybridizes to that mRNA,forming a double stranded molecule. The cell does not translate an mRNAin this double-stranded form. Therefore, antisense nucleic acidsinterfere with the expression of mRNA into protein. Preferably a DNAantisense nucleic acid is employed since such an RNA/DNA duplex is apreferred substrate for RNase H.

[0078] Oligomers of greater than about fifteen nucleotides and moleculesthat hybridize to the AUG initiation codon will be particularlyefficient. Antisense methods have been used to inhibit the expression ofmany genes in vitro (Marcus-Sekura (1988) supra; Hambor et al. (1988)Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014) and in situ (Arima et al.(1998) Antisense Nucl. Acid Drug Dev. 8:319-327; Hou et al. (1998)Antisense Nucl. Acid Drug Dev. 8:295-308).

[0079] Ribozymes are RNA molecules possessing the ability tospecifically cleave other single stranded RNA molecules in a mannersomewhat analogous to DNA restriction endonucleases. Ribozymes werediscovered from the observation that certain mRNAs have the ability toexcise their own introns. By modifying the nucleotide sequence of theseribozymes, researchers have been able to engineer molecules thatrecognize specific nucleotide sequences in an RNA molecule and cleave it(Cech (1988) JAMA 260:3030-3034; Cech (1989) Biochem. Intl. 18:7-14).Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

[0080] Investigators have identified two types of ribozymes,Tetrahymena-type and “hammerhead”-type (Haselhoff et al. (1988) Nature334:585-591). Tetrahymena-type ribozymes recognize four-base sequences,while “hammerhead”-type recognize eleven- to eighteen-base sequences.The longer the recognition sequence, the more likely it is to occurexclusively in the target mRNA species. Therefore, hammerhead-typeribozymes are preferable to Tetrahymena-type ribozymes for inactivatinga specific mRNA species, and eighteen base recognition sequences arepreferable to shorter recognition sequences.

[0081] Antisense nucleic acids and/or ribozyme can also be placedoperatively under the control of a genetic regulatory sequence of thepresent invention. Such an expression cassette, when placed in vector(e.g., a viral vector) and appropriately administered can be used toselectively modulate the expression of a particular protein in a cell,tissue or animal subject. This procedure would be complementary tomodulating the transcription of the gene encoding the protein describedabove, since in this case the gene would be transcribed but selectivelyprevented from being expressed. For example, the administration of anantisense nucleic acid or ribozyme that prevents the over-expression oftumor necrosis factor alpha, which arises in septic shock, leprosy ortuberculosis, by the methodology disclosed herein may be particularlybeneficial.

[0082] Specific Embodiments

[0083] The present invention may be better understood by reference tothe following non-limiting Examples, which are provided as exemplary ofthe invention. The following examples are presented in order to morefully illustrate the preferred embodiments of the invention. They shouldin no way be construed, however, as limiting the broad scope of theinvention.

[0084] One method for regulating the expression of a heterologous geneis to place it operatively under the control of an inducible promoter.Unfortunately, whereas promoter activity and gene expression differfundamentally when examined in vitro relative to in vivo most of theinducible promoters currently being employed have been optimized tocontrol gene expression in cultured cells, rather than in a livinganimal. For example, electrical stimulation of astroglial cells intissue culture recently has been shown to induce expression of a markergene under the control of the inducible hsp70 promoter (hsp 70) (Pelham(1982) Cell 30:517-528; Yanagida et al. (2000) J. Biotechnology79:53-61). However, the hsp70 promoter responds non-specifically to avariety of stresses including chemical toxicity, heat stress, andischemia and there was no direct evidence provided that the hsp 70promoter was specifically responsive to the electrical stimulationapplied. Moreover, whereas astroglial cells placed in tissue culture arerequired to divide, in their native state in the brain they normally donot undergo cell division. Therefore, it was not surprising to find thatin vivo the hsp 70 promoter was not effected by electrical stimulationof the brain (Example 1).

[0085] Prior to the experiments described below, the use of electricalstimulation to control the expression of a heterologous protein in vivohas not been substantiated. Therefore, a nucleic acid encoding aheterologous protein (luciferase) was placed into an expression cassetteunder the control of a genetic regulatory sequence (i.e., a 2.2 kb 5′fragment of the human glial acidic fibrillary protein gene) thatresponds to electrical stimulation. The expression cassette, therecombinant viral vector, rAAV-GFAP-Luc, was then delivered (i.e.,injected) to an animal subject. The electrical stimulation was appliedand the luciferase activity was determined in resected hippocampi.

[0086] As described in Example 2, an electrical stimulator can beinserted into the brain to control expression of a heterologous genecontained in an expression vector (e.g., a viral vector). Whereas avariety of stimulators are currently used for nerve or brain stimulationto alter neuronal firing activity, they have never been used to controlgene expression.

[0087] Example 3 describes the infusion of a peptide intranasally intorats, and subsequent changes in gene expression in a variety of brainregions were analyzed.

[0088] Parkinson's Disease is a neurodegenerative disorder characterizedby loss of the nigrostriatal pathway and is responsive to treatmentswhich facilitate dopaminergic transmission in the caudate-putamen (Yahret al. (1987) Parkinson's Disease (Raven Press); Yahr et al. (1969)Arch. Neurol. 21:343-54). Unilateral 6-hydroxydopamine lesions of thesubstantia nigra have been used to generate an established rodent modelof Parkinson's Disease.

[0089] Electrophysiology studies have demonstrated that the subthalamicnucleus (STN) has a prominent excitatory connection with the substantianigra (SN). In Parkinson's disease (PD), the subthalamic nucleus isoveractive and this overactivity may lead to progressive degeneration ofdopamine neurons in the subthalamic nucleus as well as the commonfeatures of Parkinson's disease such as tremor, rigidity andbradykinesia. As described in Example 4 below, changing the excitatoryprojection from the subthalamic nucleus to the substantia nigra into aninhibitory projection alleviates the symptoms associated withParkinson's disease.

EXAMPLES Example 1 Use of Electrical Stimulation to Regulate Genes

[0090] Materials and Methods: Rats: Male-Sprague-Dawley, Fisher, Wistarand Lewis strains of rats were used for all experiments.

[0091] Transcranial Magnetic Stimulation: An adeno-associated viral(AAV) vector expressing the luciferase transgene under control of agenetic regulatory sequence from the human 2.2 kb glial acidicfibrillary protein (GFAP) promoter was injected stereotactically intothe left dorsal hippocampus of young adult male rats. After four weeks,groups of rats were randomized to exposure to the 5 cm Cadwell coilwithout activating the stimulator (i.e., mock stimulation), to lowfrequency stimulation (5 Hz) or to high frequency (25 Hz) delivered astrans (30 trains, each 10 second duration). Stimulation or mockstimulation was given once daily for four days with the 5 cm coilcentered over the left hippocampus. On day 5, rats were euthanized andhippocampi resected and analyzed for luciferase activity.

[0092] As shown in Table 1, the amount of luciferase activity measuredcorrelated with the amount of electrical stimulation applied. As afurther control, luciferase activity in a group of completely naivecontrol rats was found to be undetectable after stimulation (<10 pg). Asis apparent from Table 1, expression of luciferase that is operativelyunder the control of the GFAP promoter element increases with electricalstimulation (see methods). Therefore, the use of 5′ elements or responseelements isolated from the promoter regions of inducible genes can beused to generate similarly regulated transcription units. TABLE 1 Effectof Electrical Stimulation on Luciferase Activity in The Hippocampi ofRats Injected with an rAAV-GFAP-Luc Vector. STIMULATION (Hz) ACTIVITY(pg/hc)^(a) FOLD INCREASE mock  109 ± 92^(b) 1  5  348 ± 178 2 25 1754 ±326 16

Example 2 Selection of Genetic Regulatory Sequences to Regulate anExogenous Gene

[0093] Bipolar stimulators were inserted bilaterally into the caudatenuclei of the rat. Stimulation was then performed on the left caudatefor 30 minutes at high frequency (200 Hz) with a current of 200 μA,while a stimulator was inserted into the right caudate but remained off.Following the stimulation the animals were sacrificed, the right andleft caudate nuclei were dissected and the tissue samples were rapidlyfrozen. mRNA was extracted from the tissues, and microarray analysis wasperformed comparing the mRNA from the left (stimulated) caudate nucleusto the right (unstimulated) caudate. This procedure was replicated infour animals.

[0094] Analysis of the data revealed significant changes inapproximately 70 genes, with a high consensus among all samples andreplicates. The majority of genes were turned off, with some genesdecreasing in amount and eight genes were expressed in increasingamounts. Promoters were next isolated by PCR from four genes which wereturned off (i.e., protein kinase B kinase, snyaptic vesicle protein 2B,phosphatidylinositol 3-kinase p85, and calcineurin B), and one gene,insulin-like growth factor-1 (IGF-1) that was increased in response tostimulation.

[0095] Next promoter fragments between 2.0 and 3.5 kb were inserted intoadeno-associated virus (AAV) vectors containing the gene for greenfluorescent protein (GFP). AAV vectors were then packaged and purifiedusing standard techniques. In a specific example, the calcineurin Bpromoter/GFP construct was packaged and 3 μl of purified vector stockswere then infused bilaterally into the rat caudate nucleus in fiveanimals. Brain stimulators were inserted bilaterally into the same pointin the brain and were fixed to the skull with dental cement. After 48-72hours of recovery, the left stimulator was turned on for 30 minutes athigh frequency (200 Hz) with a current of 200 μA, while the rightstimulator remained off. Animals were then sacrificed, and the tissuewas harvested. Protein was extracted for western blot analysis that wereperformed using a monoclonal anti-GFP protein and standard techniques.The results showed that the GFP protein levels from the left(stimulated) caudate nucleus was decreased relative to that of the right(unstimulated) caudate.

[0096] The construct was then modified to encode tyrosine hydroxylase(TH) rather than green fluorescent protein. Previously the expression oftyrosine hydroxylase from viral vectors in the caudate nucleus of ratmodels of Parkinson's disease was shown to result in therapeuticimprovement (U.S. Pat. No. 6,180,613, herein specifically incorporatedby reference in its entirety).

[0097] In an analogous experiment, the construct encoding TH was alsoshown to be responsive to electronic stimulation (i.e., as describedabove for the corresponding GFP construct). In the case of the THconstruct, a significant decrease in tyrosine hydroxylase mRNA levelsfrom the left (stimulated) caudate nucleus relative to that of the right(unstimulated) caudate was determined by quantitative polymerase chainreaction (PCR) assays.

Example 3 Use of Peripherally Administered Peptides to Regulate Genes

[0098] Peripherally Administered Peptides: A novel peptide wassynthesized that comprised nine amino acids having the amino acidsequence of HSEGTFTSD (SEQ ID NO:1). This peptide was administeredintranasally at a dose of 30 μg to rats and was found to have no effecton spontaneous locomotor behavior, stereotype, feeding, nor did itinfluence pain threshold. Twenty minutes following the administration,the rats were sacrificed and their hippocampi removed. The hippocampiwere subsequently dissected and the hippocampi RNA was isolated. Theisolated RNA was then used for gene expression profiling usingAFFYMETRIC rat gene chips (containing approximately 7000 cDNA's andEST's). Additional groups of rats received the vehicle control oramphetamine. Analysis of the gene expression data showed that incomparison to the vehicle treated rats, a large number of genes hadexpression that increased by greater than twenty fold. These genesincluded the neural adhesion molecule F3, bcl-w, MAP-2, NMDA Receptor,mGluR5 (another glutamate receptor) each of which increased by 20-70fold at this twenty minute time point.

Example 4 Administrating a Viral Vector Encoding Glutamic AcidDecarboxylase into the Subthalamic Nucleus

[0099] Electrophysiology and microdialysis were performed in thesubstantia nigra of normal rats and rats treated with a CBA-GAD65 viralvector encoding human glutamic acid decarboxylase (GAD65/67). Glutamicacid decarboxylase converts glutamate to GABA in neurons. The CBA-GADviral vector was injected into the subthalamic nucleus three weeksbefore 6-OHDA lesions of the medial forebrain bundle. Electrophysiologyand microdialysis were performed at least 4 months after thetransduction of the viral vector.

[0100] Inhibitory GABA containing connections were detected from thesubthalamic nucleus to the substantia nigra using electrophysiology andmicrodialysis. In the microdialysis experiments a much higher (˜10×)increase in GABA was detected due to low frequency electricalstimulation of the subthalamic nucleus, compared to the increase innaive control rats. Table 2 shows the extracellular concentration ofGABA and glutamate in the substantia nigra obtained before and after lowfrequency stimulation in a rat transduced with the CBA-GAD viral vectoras compared to a naive control rat. FIGS. 3 and 4 correspond to the datain Table 2. In contrast, high frequency stimulation blocked GABA releasein the microdialysis experiment, demonstrating that the release of GABA,quite independent of transcriptional regulation, can be modulated byelectromagnetic (e.g., electrical) stimulation. TABLE 2 SUBSTANTIA NIGRAMICRODLALYSIS DURING THE SUBTHALAMIC NUCLEUS STIMULATION GAD65 NAIVESample Flow Rate (1.0 GABA GLU GABA GLU 5 ul/15 ul ul/min) uM uM uM uMBasal 1 0.031 0.328 0.027 0.056 Basal 2 0.033 0.357 0.007 0.133 Basal 30.030 0.894 0.004 0.168 ST1-1 LFS-1: 10 Hz, 500 uA for 0.006 0.125 0.0040.178 2′ ST1-2 0.010 0.143 0.031 0.553 ST1-3 0.410 1.008 0.021 0.606ST1-4 0.026 0.673 0.011 0.501 ST1-5 0.139 1.290 0.032 0.644 ST1-6 0.0330.624 0.037 0.623 ST1-7 0.034 0.787 0.052 0.904 ST1-8 0.065 1.009 0.0270.514 ST1-9 0.043 0.976 0.023 0.639 ST2-1 LFS-2: 10 Hz, 500 uA for 0.0320.758 0.078 0.938 5′ ST2-2 0.023 0.819 0.108 1.121 ST2-3 0.033 0.5800.061 1.213 ST2-4 0.016 0.629 0.043 0.661 ST2-5 0.332 1.564 0.036 0.718ST2-6 0.044 0.809 0.068 1.220 ST2-7 0.049 0.863 0.049 0.796 ST2-8 0.0410.866 0.164 1.183 ST2-9 0.038 0.951 0.061 0.852

What is claimed is:
 1. A method of identifying a genetic regulatorysequence responsive to a pulsatile electromagnetic stimulus, comprising:(a) inserting a stimulator into a tissue of an animal subject; (b)applying a pulsatile electromagnetic signal with the stimulator; whereinat least a part of the tissue is stimulated; (c) identifying a gene thathas either enhanced or diminished transcription in the part of thetissue stimulated; and (d) selecting a genetic regulatory sequenceinvolved in the enhanced or diminished transcription of the gene;wherein the genetic regulatory sequence is identified as beingresponsive to the pulsatile electromagnetic stimulus.
 2. The method ofclaim 1, wherein the pulsatile electromagnetic signal is provided by abipolar stimulator.
 3. The method of claim 1, wherein the pulsatileelectromagnetic signal is provided by a monopolar stimulator.
 4. Themethod of claim 1, wherein at least a part of the tissue is notstimulated by the stimulator; and wherein the identifying of step (c) isperformed by comparing the transcription of the genes in the part of thetissue stimulated with that of a part of the tissue that is notstimulated.
 5. The method of claim 4, wherein a second stimulator isplaced into the part of the tissue that is not stimulated; and whereinno pulsatile signal is applied by the second stimulator.
 6. The methodof claim 1, wherein the tissue is neural tissue.
 7. The method of claim6, wherein the neural tissue is brain tissue.
 8. The method of claim 1,wherein the pulsatile electromagnetic signal is a magnetic stimulation.9. An isolated genetic regulatory sequence identified by performing themethod of claim
 1. 10. A vector for in vivo expression of a gene ofinterest in a mammalian host cell, wherein the vector comprises the geneof interest and a transcriptional control region comprising a geneticregulatory sequence, and wherein the gene of interest is operativelylinked to the transcriptional control region, and the genetic regulatorysequence is responsive to a pulsatile electromagnetic stimulus.
 11. Thevector of claim 10, wherein the transcriptional control region exhibitstissue specificity and is from a gene encoding a protein expressed bythe host cell.
 12. The vector of claim 10, that is a replicationdefective virus.
 13. The vector of claim 10, wherein the host cell is aneural tissue cell.
 14. The vector of claim 13, wherein the neuraltissue cell is a brain cell.
 15. A non-human mammalian host transformedwith the vector of claim 10, wherein the expression of the gene ofinterest in the non-human mammalian host can be modulated by applying apulsatile signal with a stimulator.
 16. A cell transformed with thevector of claim 10, wherein the expression of the gene of interest inthe transformed cell can be modulated by applying a pulsatile signalwith a stimulator.
 17. The cell of claim 16, wherein the transformedcell may be transplanted into a recipient mammalian host, and whereinthe expression of the gene of interest in the recipient mammalian hostcan be modulated by applying a pulsatile signal with a stimulator. 18.The replication defective viral vector of claim 10 selected from thegroup consisting of a replication defective herpes simplex virus (HSV),a replication defective papillomavirus, a replication defective EpsteinBarr virus (EBV), a replication defective adenovirus, a gutlessadenovirus, a replication defective adeno-associated virus (AAV), and areplication defective lentivirus.
 19. A method of delivering the vectorof claim 10, to a target tissue of an animal subject comprisingadministering the vector to the tissue of the animal subject, whereinthe expression of the gene of interest in the target tissue can bemodulated by applying a pulsatile signal with a stimulator.
 20. Themethod of claim 19, wherein the transcription of the gene of interest inthe target tissue is stimulated by a stimulator that is present in thetarget tissue.
 21. A method of regulating the expression of a gene ofinterest in a tissue of an animal subject in which the vector of claim10 has been administered, comprising applying a pulsatile signal with astimulator which modulates the transcription of the gene of interest inthe tissue.
 22. The method of claim 21, wherein the response to thepulsatile signal by the genetic regulatory sequence stimulates thetranscription of the gene of interest.
 23. The method of claim 21,wherein the response to the pulsatile signal by said genetic regulatorysequence hinders the transcription of the gene of interest.
 25. A methodof identifying a genetic regulatory sequence that is responsive to apeptide comprising: (a) contacting a peptide with a cell; (b)identifying a gene that exhibits altered transcription relative to acell not contacted with the peptide; and (c) selecting a geneticregulatory sequence involved in the altered transcription of the gene;wherein the genetic regulatory sequence is identified as beingresponsive to the peptide.
 26. The method of claim 25, wherein thealtered transcription is an enhancement or diminishment oftranscription.
 27. An isolated genetic regulatory sequence identified byperforming the method of claim
 25. 28. The method of claim 25, whereinidentifying a gene exhibiting altered transcription is by 2-dimensionalgel electrophoresis or Northern blot analysis.
 29. A vector for the invivo expression of a gene of interest in a mammalian host cell, whereinthe vector comprises the gene of interest and a transcriptional controlregion comprising the genetic regulatory sequence of claim 27, andwherein the gene of interest is operatively linked to saidtranscriptional control region and the genetic regulatory sequence isresponsive to the peptide.
 30. A method of ameliorating symptoms due toParkinson's disease comprising: (a) administering a vector to thesubthalamic nucleus of a patient having a symptom of Parkinson'sdisease; wherein the vector comprises a nucleic acid encoding glutamicacid decarboxylase operatively under the control of a genetic regulatorysequence that is stimulated by a pulsatile electromagnetic signal; (b)inserting a stimulator into the subthalamic nucleus of the patient; and(c) applying a pulsatile electromagnetic signal with the stimulator,wherein the glutamic acid decarboxylase is expressed and leads to theamelioration of a symptom of Parkinson's disease.
 31. The method ofclaim 30, wherein the vector is a replication defective viral vector.32. The method of claim 30, wherein the vector is a CBA-GAD65 viralvector.
 33. A method of modulating the release of a stored compound by acell comprising: (a) administering a vector to the subthalamic nucleusof an animal subject; wherein the vector comprises a nucleic acidencoding a protein operatively under the control of a genetic regulatorysequence that is stimulated by a pulsatile electromagnetic signal, andwherein the expression of the protein stimulates the production of acompound that is stored by a cell of the animal subject; (b) inserting astimulator into the subthalamic nucleus of the animal subject; (c)applying a pulsatile electromagnetic signal with the stimulator, whereinthe protein is expressed and the compound is produced; and wherein thecell stores the compound; and (d) applying a second pulsatileelectromagnetic signal with the stimulator, wherein the release of thestored compound from the cell is modulated.
 34. The method of claim 33,wherein the compound is GABA.
 35. The method of claim 34, whereinapplying of a second pulsatile electromagnetic signal is at a lowfrequency and the release of GABA from the cell is increased.
 36. Themethod of claim 34, wherein applying of a second pulsatileelectromagnetic signal is at a high frequency and the release of GABAfrom the cell is decreased.